Support is requested to develop new methods for the analysis of important biological constituents through the use of immobilized enzyme technology. These methods will involve the use of specific enzyme reactions either as an on-line preparative step prior to a detection system for separations carried out by high performance liquid chromotography. This combination should be synergistic since the separation step can be used to eliminate interferences which might be present in the sample and would deteriorate the quality of the assay. In contradistinction, the use of class specific enzyme reactions will allow detection of only the species of interest, thus avoiding either pre-chromatographic clean up or development of a complex separation system. It may also provide a specific derivatization technique for species which do not absorb significantly in the normally used portion of the UV spectrum. The use of immobilized enzymes will be studied in the presence of organic solvents to facilitate coupling of the enzyme reactors to reverse phase liquid chromatography. The specific projects for which support is requested are: a) the use of an immobilized enzyme reactor to achieve post column derivatization as a means of detection of bile acids and their conjugates using 3 alpha-hydroxysteroid: NAD oxidoreductase (E.C. 1.1.1.50) after separation by reverse phase HPLC; and b) the rapid equilibrium hydrolysis of glucuronide conjugates as an on-line preparative step prior to the reverse phase HPLC separation of estrogens and adrenogenital steroids.